Melampyrum sylvaticum is an endangered annual hemiparasitic plant that is found in only 19 small and isolated populations in the United Kingdom (UK). To evaluate the genetic consequences of this patchy distribution we compared levels of diversity, inbreeding and differentiation from ten populations from the UK with eight relatively large populations from Sweden and Norway where the species is more continuously distributed. We demonstrate that in both the UK and Scandinavia, the species is highly inbreeding (global F IS = 0.899). Levels of population differentiation were high (F’ST = 0.892) and significantly higher amongst UK populations (F’ST = 0.949) than Scandinavian populations (F’ST = 0.762; P < 0.01). The isolated populations in the UK have, on average, lower genetic diversity (allelic richness, proportion of loci that are polymorphic, gene diversity) than Scandinavian populations, and this diversity difference is associated with the smaller census size and population area of UK populations. From a conservation perspective, the naturally inbreeding nature of the species may buffer the species against immediate effects of inbreeding depression, but the markedly lower levels of genetic diversity in UK populations may represent a genetic constraint to evolutionary change. In addition, the high levels of population differentiation suggest that gene flow among populations will not be effective at replenishing lost variation. We thus recommend supporting in situ conservation management with ex situ populations and human-mediated seed dispersal among selected populations in the UK.